Project Summary/Abstract Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the Huntingtin (HTT) protein. A hallmark of HD found in the brains of patients is the presence of intracellular deposits containing the N- terminal fragment of mutant HTT (mHTT) with an expanded poly-glutamine sequence. Mutant HTT is prone to misfold and form aberrant species, including oligomers, fibrils and bundles of fibrils; one or more of these species can be toxic, particularly to neurons. Mutant HTT species is modulated by the protein homeostasis (or proteostasis) status of the cell. The 1 MDa, ring-shaped hetero-oligomeric chaperonin TRiC/CCT, essential for proteostasis, has emerged as a central regulator of mHTT aggregation and toxicity. TRiC is a chaperone that. Our preliminary data showed that TRiC binds to wild type (WT) and mHTT monomers, oligomers and fibers suppressing both aggregation and toxicity. Further, we find that overexpression of TRiC or small TRiC-derived domains remodels mHTT aggregates in vivo and in vitro and can protect from mHTT toxicity in HD models. We hypothesize that understanding the action of TRiC on mHTT can provide a therapeutic avenue for HD treatment. This Project will provide a deeper mechanistic and structural understanding of how TRiC and TRiC- derived domains (hereafter TRiC reagents) interact with mHTT species at the different stages of the aggregation pathway, including monomers, oligomers and fibrils, and how they influence the misfolding and aggregation landscape of mHTT. Aim 1 will use biochemical, functional and cryo-electron tomographic analyses to characterize mHTT aberrant species and their interactions with TRiC reagents using recombinant proteins. Aim 2 will examine the effects of TRiC reagents on the formation and remodeling of mHTT aberrant species in the cellular context, using neuronal lysates from HD cell models, including primary neurons from R6/2 and BACHD mice. Aim 3 will use structural and biochemical assays to examine how mHTT expression affects selected subcellular components such as axonal microtubules and mitochondria, and TRiC proteostasis in HD cell models with and without TRiC reagent treatment. We will establish a scoring system indicative of the effects of the presence of TRiC reagents on our biochemical and structural assays of mHTT species both ex vivo and in vitro. Our experiments are highly synergistic with Projects 2 and 3, which assess TRiC's role in ameliorating pathogenesis in HD neuronal and HD animal models. The results from this Project will provide a conceptual framework for these more physiological and translational studies by defining new, optimized TRiC reagents and which cellular loci and processes to examine, in preparation for engaging in studies that could establish proof-of-concept TRiC-based therapeutics for Huntington's disease.